Small size gene analysis apparatus

ABSTRACT

By the conventional technique for dispensing more than one reagents accurately, the system is complicated and thus a compact and inexpensive system is difficult to realize. In the present invention, the pressurized dispensing system utilizing a capillary is realized, and in addition, in order to reduce the leakage of reagents different from the reagent dispensed, by forming air layers at the tips of the capillaries after dispensing, a compact, simple, inexpensive analysis apparatus is realized.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to an apparatus analyzing nucleic acids,and in particular, an apparatus capable of analyzing gene sequences,gene polymorphism, and gene mutation.

(2) Description of Related Art

For determining DNA base sequences, methods using gel electrophoresisand fluorescence detection are widely used. In such method, first, manycopies of a DNA fragment are made, the sequence of which is to beanalyzed. Fluorescence-labeled fragments of various lengths areprepared, 5′-terminals of the DNA being starting points, whereinfluorescent labels are also attached, wavelengths varying with bases at3′-terminals of these DNA fragments. The difference in length isidentified by one base by gel electrophoresis, and emission from each ofthe fragment groups is detected. DNA terminal base types of the DNAfragment groups being studied are elucidated according to colors ofemission wavelengths. The DNA fragment groups pass through thefluorescent detection section one by one from a shorter one, so thatterminal base types can be identified consecutively from a shorter DNAby measuring fluorescence colors. Thereby, the sequence is determined.Such fluorescent DNA sequencers are widespread, and also theircontribution to the Human Genome Project was enormous. On the otherhand, the Human Genome Project was completed, as declared in 2003, andthe time has come to make use of sequence information in medicine andvarious industries. There, in many cases, analysis of entire long DNA isnot required and elucidation of a short DNA sequence of interest issufficient. For such DNA sequence analysis, simple methods andapparatuses are required.

The sequence determination by stepwise chemical reactions such aspyrosequencing is a technique developed in order to meet suchrequirement (for example, Patent document 1 and Patent document 2). Inthis method, a primer is hybridized with a target DNA strand, and fournucleic acid substrates for complementary strand synthesis (dATP, dCTP,dGTP, and dTTP) are added one by one in order into the reactionsolution, and thereby a complementary strand is synthesized. Uponcomplementary strand synthesis, as the complementary DNA strand extends,pyrophosphate (PPi) is generated as a by-product. In the presence of anenzyme, pyrophosphate is converted to ATP, which in turn goes throughthe reaction in the presence of luciferin and luciferase to generateemission. By detecting this emission, the incorporation of the nucleicacid substrates for complementary strand synthesis in the DNA strand isconfirmed, and the sequence information of the complementary strand, andconsequently the sequence information of the target DNA strand will beelucidated. On the other hand, the nucleic acid substrates forcomplementary strand synthesis that have not been used in the reactionare promptly degraded by an enzyme such as apyrase so as not tointerfere with subsequent reaction steps (for example, Patent document2). Many apparatuses for this pyrosequencing employ chemiluminescentdetection system, wherein a titer plate having 96 reaction cells (havinga volume of 100 μl or less) is utilized as a reaction cell plate. Insuch apparatus, each of the four nucleic acid substrates forcomplementary strand synthesis (dATP, dCTP, dGTP, and dTTP) is containedin a separate reagent vessel and injected into the reaction cells one byone (for example, Patent document 3). That is, DNA, a primer, enzymesfor synthesizing a complementary strand, chemiluminescent reagents, andthe like are placed in advance in the reaction cells; a reagentdispenser comprises four nozzles; the nozzles or a titer plate is movedin the x-y directions as well as in the rotation direction; the air inthe reagent vessels is pressurized; and thereby the reagents are drippedone by one from the tips of the nozzles, thus emission being detected.

Furthermore, a technology to provide a small size apparatus for theabove pyrosequencing is disclosed (for example, Patent document 4). Inthis technology, a narrow tube is connected from each of dNTP vessels tothe reaction section; it is suggested that compact and simple analysisis attainable by the method wherein four dNTPs are injected one by oneby using these narrow tubes.

On the other hand, a luminescence detection apparatus utilizing apressurized dispensing system for dispensing reagents is disclosed as asmall size apparatus for measuring bioluminescence (for example, Patentdocument 5). In this technology, capillaries for dispensing are alignedwith reaction cells one by one, and dispensing reagents is controlled bypressurization.

Moreover, in regarding to reagents that can be used for thepyrosequencing reaction, an example of a reaction system different fromthe technologies described above is disclosed (for example, Patentdocument 6). In this conventional technology, AMP and PPi aresynthesized into ATP by using the reverse reaction of the enzyme,pyruvate, phosphate dikinase (PPDK), and AMP concentrations aremeasured.

Patent document 1: WO 98/13523

Patent document 2: WO 98/28440

Patent document 3: WO 00/56455

Patent document 4: JP-A-2001-258543

Patent document 5: JP-A-2004-12411

Patent document 6: JP-A-9-234099

It is believed that because the reaction mechanism used is simple, thepyrosequencing method is suitable for small size and inexpensiveapparatuses. Four nucleic acid substrates for complementary strandsynthesis are required for measurement, as described above, and hencethese need to be measured accurately. In order to make an apparatussmall and inexpensive, it is also essential to design to use a minutetotal amount of reagents.

In the conventional technology, there is a problem that an accuratereagent dispensing mechanism can not be small and inexpensive. Forexample, in order to make an apparatus small, dispensing about 0.1 to0.2 μl needs to be performed within an error of 10% or less. However,conventionally, in the method of dripping reagents, which is said to bea simple method of dispensing reagents, for example, on dispensing 0.4μl, a dispensing error of about 15% occurs, and on dispensing less than0.4 μl, in many cases, dispensing is not possible due to surface tensionof the liquid. Furthermore, another example to realize micro-dispensingis the Bubble Jet® technology, in general, used for inkjet printers,which has problems such that reagents are deteriorated by heating andthat it is difficult to simplify replenishment and maintenance.Moreover, in the pressurized dispenser method using capillaries, whichcan realize simple, inexpensive, accurate dispensing, nevertheless,because the tip of the capillary is in contact with a sample solution inthe reaction vessel, reagents may disadvantageously leak at the time theair is not pressurized.

Furthermore, four reagents need to be injected into a reaction vessel ina predetermined order. In the conventional nozzle method, there areproblems that miniaturization is difficult and parallel arrangement isalso difficult. That is, in a 96-well titer plate widely used in thisfield, 96 reaction vessels (holes) are placed with a pitch of 9 mm, butit is impossible to provide a plurality of nozzles with a pitch of 9 mmby the conventional technology. Therefore, the reagents are dispensedthrough a set of nozzles into multiple reaction vessels, so that themeasurement efficiency is low as well as the horizontal mechanismmovement tends to be large and expensive.

Furthermore, the mechanism that allows the dispensed substrates to admixwith a sample in the reaction vessel efficiently is required. In orderto realize a simple, small size, inexpensive apparatus, these problemsshould be solved.

SUMMARY OF THE INVENTION

In order to solve the problems described above, a single-piecedispensing chip having four reagent containing spaces has been inventedin the present invention. This chip employs a pressurized dispensingsystem utilizing capillaries and having a high dispensing accuracy. Inorder to realize placement with a pitch of 9 mm, the chip isminiaturized, and will be attached to the head so that replenishment ofthe reagents, etc. can be simplified. The chip is disposable, designedto be used up.

Furthermore, a vertical movement mechanism is provided on the head partholding the chip. Thereby, whether the capillary and the liquid in thereaction cell are in contact or not can be controlled at the time ofreagent dispensing and at the time of stirring. Furthermore, an air gapis provided in the capillary. Thereby, a reagent is kept from leakingfrom the tip of the capillary of the chip. As compact and simple meansof forming an air gap, a microejector utilizing a high pressure gas(such as air or nitrogen) operated by the pressurized dispensing systemis provided, and the negative pressure generated thereby is utilized.Consequently, the formation of an air gap can be reliable.

An example of the analysis apparatus in accordance with the presentinvention is characterized in that the analysis apparatus comprises:reagent container-holding means for holding a reagent containercontaining a reagent; moving means for moving the reagentcontainer-holding means vertically; a reaction vessel for receiving asupply of the reagent from the reagent container and containing theliquid; pressurizing means for applying pressure to the reagentcontainer to supply the reagent therefrom to the reaction vessel;vibrating means for applying vibration to the reaction vessel; and adetector for optical detection for the reaction vessel.

Another example of the analysis apparatus in accordance with the presentinvention is characterized in that the analysis apparatus comprises:reagent container-holding means for holding a reagent containercomprising a reagent delivering part and containing a reagent; movingmeans for moving the reagent container-holding means vertically; areaction vessel for receiving a supply of the reagent from the reagentcontainer and containing a sample; pressurizing means for applyingpressure to the reagent container to supply the reagent therefrom to thereaction vessel; vibrating means for applying vibration to the reactionvessel; a detector for optical detection for the reaction vessel; and anegative pressure-generating mean for providing an air layer inside thereagent delivering part.

An example of the reagent kit in accordance with the present inventionis characterized in that the kit includes: a first vessel, whichcomprises a first liquid delivering part and contains a first liquid; asecond vessel, which comprises a second liquid delivering part andcontains a second liquid; a third vessel, which comprises a third liquiddelivering part and contains a third liquid; and a fourth vessel, whichcomprises a fourth liquid delivering part and contains a fourth liquid,and that the first liquid delivering part, the second liquid deliveringpart, the third liquid delivering part, and the fourth liquid deliveringpart are essentially placed being symmetric with respect to a point.

The present invention can realize a small size and inexpensive apparatusthat analyzes nucleic acids and gene sequences. First, by the method inaccordance with the present invention, dispensing chips as many asdesired can be inexpensively arranged in parallel. Furthermore, desireddispensing accuracy can be attained in a compact and simple manner. Inaddition, replenishment of the reagents, etc., is easy. Moreover, thepresent method is suitable for admixing of the reagent that determinesreaction efficiency. Thereby, sequence analysis can be performed withhigh accuracy. Furthermore, by preventing a pressurized dispensingsystem utilizing capillaries from leaking a reagent, accuracy of thereaction can be improved.

Other objects, features and advantages of the invention will becomeapparent from the following description of the embodiments of theinvention taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIGS. 1(a) and 1 (b) illustrate examples of the configuration of theapparatus;

FIG. 2 illustrates the dispensing chip;

FIGS. 3(1), 3(2), and 3(3) are illustrations to explain the dispensinghead into which the chips are inserted;

FIG. 4 illustrates a sequence to explain the operations of the apparatusat the time of measurement;

FIG. 5 illustrates the results of sequence analysis;

FIG. 6 illustrates a sequence of the operations of the apparatus at thetime of measurement;

FIG. 7 graphically shows a relation between the amount of ATP dispensedand the level of luminescence;

FIGS. 8(a) and 8(b) illustrate a microejector and an example of theconfiguration of the apparatus including the microejector, respectively;and

FIG. 9 illustrates the levels of luminescence when two differentconcentrations of the reagent are dispensed twice each.

FIG. 10 shows another example of a sectional view of a microejector.

DETAILED DESCRIPTION OF THE INVENTION Embodiment 1

Now, the present invention will be described referring to Embodiments.

In the present invention, the target gene sequence to measure isdetermined by using the principle of the pyrosequencing method describedin the Description of Related Art section. First, an example of theconfiguration of the apparatus for analysis according to the presentinvention is shown in FIG. 1(a). First, the apparatus has a reactionvessel holder 101. The holder 101 supports four reaction vessels 1011 to1014 in a single row with a pitch of 9 mm. The present apparatusperforms nucleic acid extension using enzymes, and enzymatic reactions,in general, work efficiently when the temperature is higher than roomtemperature, so that it is more preferable that temperature controllingmeans (such as a peltier element), by which heating or cooling to agiven temperature can be conducted, is connected to the holder. Inaddition, in this embodiment, four reaction vessels are placed with apitch of 9 mm (a pitch of a 96-hole titer plate), but any number ofreaction vessels and any pitch can be given in applications and thenumber of reaction vessels and the pitch size are not limited. Moreover,the total number of reaction vessels can be increased by providing morethan one row shown in FIG. 1. The holder 101 can be vibrated in itsentirety by a vibration generating device such as a vibration motor.Such vibration is useful for admixing dispensed reagents and a sample ina reaction vessel upon dispensing reagents.

A light detection part 102 is covered in its entirety by a casing madeof an electrically conductive material, having four photodiodes facingtoward the reaction vessels in alignment with the pitch of the reactionvessels. The interface with the reaction vessels has a glass 1021 havinga transparent electrode layer (ITO, etc.) at the back. This transparentelectrode is electrically connected to the electrically conductivecasing covering the entirety, and is connected to ground potential ofthe apparatus. In addition, inside the casing, an amplifier thatamplifies signals from the photodiodes is included, the amplifierconnecting to an A/D conversion circuit outside the detection part.

The A/D conversion circuit 103 digitizes light detection signals andtransmits data to a computer for controlling the apparatus and forreceiving the data.

A dispensing head 104 has a function as means of holding dispensingchips, by which dispensing chips are held therein. Herein, the chipcomprises capillaries for dispensing, the chip being held in a mannerthat the end of the capillary for dispensing opposes the reactionvessel. Furthermore, a group of four air tubes for pressurization 105,each of which corresponds to one of four reagents, respectively, areconnected to a group of solenoid valves 106. However, the dispensinghead and the group of solenoid valves may be made as a single piece. Inaddition, a mechanism for moving up and down 107 the entirety of thedispensing head is included. In regard to the vertical movementmechanism 107, for example, it is easy to provide a rack 1071 to thehead 104 and drive a pinion 1072 by a motor, etc., as shown in FIG.1(b). In such case, the vertical position of the head can be controlledwith a contact switch or by the number of revolution of the motor.Moreover, the effects that can be obtained when the vertical positioncan be controlled will be described later. To put it simply, it is alsopossible to move the head up and down by using a syringe that utilizesgas provided from a high pressure gas source. Such method is simple,because controlling up and down movements is attained by filling the gaswith the use of a single solenoid valve.

The group of solenoid valves have four 3-way solenoid valves, and itsside of a high pressure gas source is connected to a high pressure gastank included in the apparatus or a high pressure gas line sourceprovided in a laboratory. Furthermore, its exhaust side is connected viaanother solenoid valve 108 to a microejector, which is a negativepressure source 109. The negative pressure source 109 uses themicroejector and the high pressure gas source to generate negativepressure of about 0.5 atm. The generation of negative pressure iscontrolled by the solenoid valve 108.

A top view and a sectional view of the dispensing chip are shown in FIG.2. The dispensing chip includes reagent vessels 201 to 204 containingeach of four reagents, respectively. Each of the reagent vesselsincludes a capillary 2011 to 2014, respectively, the capillary being adispensing path. In this embodiment, a glass capillary, the total lengthbeing 20 mm, the outer diameter being about 350 μm, and the innerdiameter being about 50 μm, is used as a capillary. The inner diameterand the length of a reagent vessel are 2.4 mm and 10 mm, respectively.The volume of a reagent vessel is about 45 μL. Four reagents containedin the reagent vessels are expected to be deoxynucleotide triphosphates(dNTPs), for example. Particularly, in each of the four reagent vessels,deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP),deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate(dGTP) are expected to be contained. Moreover, analogues of dNTPs mayalso be used. For example, in place of triphosphate, dNTPαS, wherein onephosphorus at the α-position is substituted with a sulfur, and the likemay be used.

In the dispensing chip, the four reagent vessels are essentially placedbeing symmetric with respect to a point, so that the location where eachreagent is dispensed is equally placed from the center. There are thefollowing advantages. First, by coinciding the center of the dispensingchip with the center of the reaction vessel, no location differencearises among the four reagents. That is, the location in the reactionvessel of a capillary is closely related to the degree of admixing of areagent after dispensing. The reaction vessel is a cylinder, and hencethe ideal location for dispensing is the center of the reaction vessel,when the effect of admixing a reagent is considered. However, when thefour reagents are dispensed as a package, and if all are to be at thecenter, a horizontal mechanism movement and the like are required,resulting in an increase in cost of the apparatus. Therefore, in orderto handle the four reagents equally, the placement that is essentiallyconcentrical from the center of a reaction vessel is most suitable.

Furthermore, the bottom of the reagent vessel, i.e., the connection partwith the capillary, has essentially a conical shape (2021), the centerresiding at the location where the capillary is placed. Thereby, theamount of a reagent which becomes undispensable, i.e., the dead volumecan be minimized. Moreover, the dispensing chip itself is symmetric withrespect to a line, and it is convenient to provide a pin for positioning2022 in order not to misplace the four reagents. Moreover, the end ofthe top of the chip 2023 is designed to be essentially on the sameplane. Thereby, it is advantageously easy to secure airtightly the chipsin a chip holder by airtight members 3001 to 3004, which will bedescribed later. Likewise, it is also easy to apply to the top a lid forsealing or a sealing agent so as to prevent the reagents from drying.

Furthermore, this chip itself is a cylinder having a diameter of lessthan 9 mm, and hence it can be effectively placed at the titer plate'spitch.

FIG. 3 is a schematic illustration of the dispensing head. (1) is abottom view of the top of the dispensing head, (2) is a sectional viewof the head, and (3) is a top view of the bottom of the dispensing head.The dispensing head is assembled in a manner that the bottom of (1) andthe top of (3) are joined. More particularly, the dispensing head can beseparated into the bottom 301 of the dispensing head and the top 302 ofthe dispensing head, the dispensing chip shown in FIG. 2 is insertedtherebetween. To the bottom of the dispensing head, a hole to allow thecapillaries of each dispensing chip to go through is provided. To thetop of the dispensing head, four high pressure gas flow entrances perchip, which are individually connected to each of the reagent vessels ofthe chip, are provided. The part 303 refers to one of high pressure gasflow paths. One flow path of the high pressure gas corresponds to onereagent vessel, and is connected to the gas flow entrance of eachdispensing chip. The flow paths gather into one, which is connected toone air tube. The airtight members 3001 to 3004, being in tight contactwith the chips, can keep each reagent vessel in an airtight condition.

For a member most suitable to provide air tightness, materials havingrubber elasticity such as silicone rubber and viton® rubber aresuitable.

First, characteristics of the dispensing chip and others in accordancewith the present invention are described. The pressurized dispensingsystem utilizing capillaries is characterized by high dispensingaccuracy on microdispensing. For example, in this embodiment, adispensing error of about no more than 10% was attained on dispensing ata dispensing volume of 0.2 μl. Furthermore, with smaller capillariesthan the one having an inner diameter of 25 μm in this embodiment, adispensing error was no more than 8%.

Here, the effect that can be obtained by using narrow tubes(capillaries) is described. In the dispensing method in accordance withthe present invention, the volume Q of the reagent dispensed isdetermined by the pressure applied from outside and the duration ofpressurization according to the following Hagen-Poiseuille law.Q=ΔP·π·r ⁴ ·t/(8 μL)wherein ΔP is the pressure, r is the radius of a capillary, t is theduration of pressurization, μis the viscosity of a solution, and L isthe length of a capillary.

Dispensing accuracy depends on controllable mechanisms, and hence inthis case evaluations of an error of the pressure applied and an errorof the duration of pressurization are important. As shown in the aboveequation, when these errors are the same, the error of the dispensedvolume caused by such errors is proportional to the fourth power of theradius of a capillary, and is inversely proportional to the length of acapillary. Therefore, the smaller the radius, the smaller the influenceof the error of the pressure applied and the error of the duration ofpressurization on the dispensing error. In this embodiment, capillariesin the range of from 50 μm to 25 μm (inclusive) in diameter are used.Capillaries having a diameter of about 1 to 25 μm may be used but tendto be clogged depending on reagents to be dispensed. On the other hand,with capillaries having a diameter of more than 50 μm, as describedabove, dispensing accuracy is deteriorated. The capillary diameter foruse can be selected, as needed, according to a dispensing volume in themode in use and required accuracy, but under the conditions in thisembodiment wherein a dispensing volume and clogs are considered,capillaries having a diameter of from 25 to 50 μm (inclusive) aresuitable. A dispensed volume was determined from a change of massbetween before and after dispensing, and the error from this was closeto the measurement limit. Furthermore, in this system, by allowing thetips of the capillaries to be kept in contact with the liquid in thereaction vessel even when dispensing is not performed, the verticalmovement mechanism 107 could be eliminated. However, when the tips ofthe capillaries are kept in contact with the liquid in the reactionvessel, reagent leakage, which should be avoided, from the tips of thecapillaries is anticipated. That is, in ordinary use, the inside of acapillary is filled with a reagent. In this apparatus, it is necessaryto dispense any of the four reagents in any volume on nucleic acidanalysis, and hence unexpected leak of a reagent into the reactionvessel causes an unexpected reaction to occur, resulting in a seriousmeasurement error. Therefore, the volume of leakage by diffusion of areagent in this apparatus was evaluated below. The evaluation wasperformed by using bioluminescent reagents. First, into a reactionvessel, bioluminescent reagents, luciferase and luciferin, having beendissolved in a buffer were added, and into a capillary, ATP was added asa reagent. As shown in FIG. 7, the level of luminescence to the amountof ATP dispensed is linear and known. Thus, an amount of leakage of ATPcan be evaluated by measuring weak luminescence, when dispensing is notperformed, i.e., the high pressure gas is not injected, the capillarybeing set as it is.

First, the reaction vessel was kept still, and the volume of leakage fortwo min was measured, but no leakage was observed. On the other hand, inorder to stir the reagent, the reaction vessel was vibrated by avibration generating device for 20 sec, and a large amount of leakagewas observed. That is, when the reagent was stirred, it was found thatit is important to separate the tip of the capillary from the reagentbefore stirring. However, in case that the vertical movement mechanismwas used, when the tip of the capillary was inserted back again into theliquid in the reaction vessel, reagent leakage that may be caused by theimpact thereof was observed. For example, with a capillary having aninner diameter of 50 μm, by the impact arising when the tip was insertedinto the surface of the liquid in the reaction vessel, reagent leakageof about 6 nL was observed. And, likewise, with a capillary having aninner diameter of 25 μm, reagent leakage of about 3 nL was observed.These problems can be solved in a manner that after dispensing thereagent, the tip of the capillary is separated from the surface of theliquid, and then a layer of gas (air in this case), i.e., the air gap,is formed at the tip of the capillary (the end where liquid isreleased). This is because the presence of the layer of gas causes thelocation of the boundary of the reagent inside the capillary to retractby about 5 mm, so that accidental leakage from the tip of the capillarycan be avoided. The air gap, in general, can be formed by sucking thereagent vessel using a syringe. However, when a syringe is used,disadvantages arise because the system becomes costly and complicatedincluding the mechanism related to the syringe. Therefore, in thisembodiment, in order to provide a simpler method, negative pressure byusing a microejector is utilized. A microejector, as shown in asectional view of FIG. 8(a), is means of generating negative pressurethat comprises a narrow tube 81 and a housing 82 linked to the highpressure gas source. When the high pressure gas flows into themicroejector from a flow entrance 801 to an exhaust slot 802, a flowlike 803 occurs in the vicinity of the narrow tube residing inside, andthereby the air inside the narrow tube is retracted in the direction of804, resulting in the generation of negative pressure in the directionof 805. This apparatus already includes the high pressure gas source,and hence by using the microejector, negative pressure can be readilyobtained. The reagent vessel is connected to the microejector for veryshort duration and thereby negative pressure is applied to thecapillary, thus negative pressure being generated. As a result, the airgap of about 5 mm contributed to the prevention of reagent leakage, andwith the capillary having an inner diameter of 50 μm, the volume ofleakage could be reduced to about 0.6 nL. The system to generatenegative pressure by using the microejector is very compact and simple,and is suitable for apparatuses for analyzing nucleic acids such as forexamining genes on a small scale. An example of the constitutioncomprising a microejector is shown in FIG. 8(b). Herein, a microejector1000 comprises a narrow tube 81 and a housing 82. The housing 82 isdirectly connected to the high pressure gas source via a solenoid valve108 and a tube 810. Furthermore, the narrow tube 82 is connected to theexhaust slot of the group of solenoid valves 106 by the tube 811. Whenthe gas flows from the high pressure gas source via the tube 810 intothe housing 82, negative pressure is generated in the narrow tube 81, asdescribed above, to suck the exhaust slot of the group of solenoidvalves 106. The group of solenoid valves 106 are 3-way, as describedabove, and hence when the valves are OFF, the exhaust slot is directlyconnected to a group of tubes 105. Therefore, sucking the exhaust slotnamely means sucking the air in the reagent vessels in the dispensingchip. Because the duration of sucking can be controlled by ON/OFF of thesolenoid valve 108, the air in the reagent vessels can be sucked fordesired duration. Accordingly, when the duration of sucking is suitablypredetermined, a desired air gap can be formed at the tip of thecapillary.

FIG. 10 shows another example of a sectional view of a microejector. Itcomprises a tube 1101, a housing 1100 and a tube 1102. The tube 1101 isconnected to the high pressure gas source and the tube 1102 is connectedto the regent vessels. When the high pressure gas flows into themicroejector from a flow entrance like 1111 to an exhaust slot 1112, aflow like 1113 occurs in surroundings in the narrow tube end, andthereby the negative pressure is generated like 1114.

The problem of reagent leakage is very important in apparatusesanalyzing nucleic acids. For example, in sequence analysis utilizingpyrosequencing, one of four nucleic acid substrates is dispensed, andwhether extension occurs thereby or not is confirmed by luminescence;therefore, if a nucleic acid substrate different from the one ofinterest is intermixed, it directly becomes an analysis error. Whenevaluating a plurality of base sequences continuously, the analysiserror exponentially increases with the number of sequences, so that thelength of bases that can be analyzed is extremely limited. Therefore,reducing the volume of reagent leakage is an important objective. In theapparatus for analyzing nucleic acids in this embodiment, a reactionvessel uses about 20 μL of a sample solution. Compared with that, 0.6 nLcorresponds to no more than 1/10000, and this is negligible. That is, bythis constitution, the volume of leakage can be decreased to thenegligible level.

Next, the method for analyzing genes using the apparatus of thisembodiment is described. FIG. 4 is an illustration of the time sequenceof operations of the dispensing head, pressurization, and the vibrationstirring motor, when one of the four dNTPs (i.e., dATPαS, dGTP, dCTP,and dTTP) is used as a reagent and is dispensed from the dispensing chipof this embodiment. The operations are as follows. First, the dispensinghead is lowered so that the surface of the liquid in the reaction vesselbecomes in contact with the tip of the capillary. Next, for the durationcorresponding to a desired volume of the reagent to be dispensed,pressurization is performed by the high pressure gas to dispense thereagent. Then, the dispensing head is raised, and thereby the tip of thecapillary is separated from the surface of the liquid in the reactionvessel. Finally, the stirring motor is operated for a predeterminedperiod of stirring, and at the same time, sucking for negative pressureis performed for predetermined duration in order to form the air gapinside the capillary. For analyzing gene sequences, such series ofoperations can be repeated for every base. An example of the genesequence analysis obtained in this embodiment is shown in FIG. 5. There,in FIG. 5, the substrates, A, G, C, and T refer to dATPαS, dGTP, dCTP,and dTTP, respectively. That is, an analogue of dATP is used for A,only. It is noted that the results of the analysis completely coincidewith the base sequence of the sample, which has been already known.

Embodiment 2

Another embodiment related to dispensing reagents using the apparatusdescribed in Embodiment 1 is described. First, when one substrate isdispensed, a lower concentration or a smaller volume of the reagent ispreliminarily prepared and dispensed plural times. By using the presentapparatus, such dispensing method can be readily realized. This iseffective on occasions such that a concentration of the reagent islowered in order to decrease leaking molecular weight, when the volumeof leakage is about 0.6 nL, as described above; or that on continuoussequence analysis, a sequence continuously formed by the same base isobserved, and thus the need of the additional reagent arises. In thisembodiment, a case wherein dispensing is performed twice is described.FIG. 6 is an illustration of the time sequence of operations of thedispensing head, pressurization, and the vibration stirring motor, whenone reagent is divided into two portions to dispense. When a singlereagent is divided into two portions and dispensed, the emissionfollowing the first dispensing and the emission following the seconddispensing are compared, and thereby whether the extension of thenucleic acid sample is completed or not can be determined. FIG. 9 is anexample wherein by using different concentrations, dispensing wasperformed twice. The dashed line refers to the experiment using a ½concentration of the concentration of the reagent dispensed in theexperiment expressed by the solid line. In both experiments, dispensingwas performed twice at 901 and 902. This figure indicates that when theinjected reagent was sufficient, no emission was observed following thedispensing at 902, whereas with the ½ reagent concentration, componentsthat had not reacted remained, so that further emission was observedfollowing the second dispensing. This experimental example presents oneexample, and likewise, by injecting multiple times to obtain emissionsignals thereby, whether the reagent injected is sufficient or not canbe determined.

This is effective to improve accuracy of sequence analysis, because aninjecting amount of a reagent should be the optimum amount for thereaction. If the injecting amount is insufficient, nucleic acids thathave not reacted will cause noises. On the other hand, when an excessiveamount has been added and the next reagent is injected, the previousreagent that has not been completely degraded remains, resulting in acarry-over. A carry-over causes backward reading, etc., deterioratingmeasurement accuracy.

The optimum amount of a reagent varies with the number of basesextending. That is, when extending by two bases, the reagent twice inamount is required compared with when extending by one base. In manycases, the number of bases of the nucleic acid sequence of interest isunknown, and hence the number of bases extended by the injected reagentis not known. Thus, the optimum amount of the reagent can not be decidedin advance. In such case, dispensing multiple times is effective.

Embodiment 3

Another embodiment related to the method of dispensing a plurality ofreagents simultaneously using the apparatus described in Embodiment 1 isdescribed.

In Embodiment 2, four substrates are dispensed one by one, but two,three, or all, i.e., two or more substrates can be optionally dispensedat the same time. It is important that the same dispensing chip that isused for usual sequence analysis can be used in such case as well. Forexample, in one of the techniques of analyzing polymorphism, in order toevaluate polymorphism at the extension probe side, polymorphism isallowed to be present at the 3′-terminal, wherein complementarity isanalyzed. In this case, a mixture solution of four substrates is used asthe reagent. Therefore, handling is different from normal reagents forsequence analysis, and a mixture solution of four substrates isparticularly prepared for the polymorphism analysis. However, in thepresent apparatus, after sequence analysis is conducted, the samereagent dispensing tubes can be used only by changing samples inreaction vessels. Accordingly, various types of analysis can be simplyattained.

In addition, when a known sequence is analyzed for confirmation and thepossibility of heterogeneous SNPs is expected in advance, by dispensingsuch two bases simultaneously, a phase shift disadvantageously arisingin pyrosequencing can be eliminated.

Embodiment 4

In regard to the dispensing chip described in Embodiment 1, anotherembodiment related to supplying measures to investigators is described.Herein, there is an example wherein simpler reagent management isprovided for investigators as well as for suppliers. In the presentinvention, four nucleic acid substrates are used by injecting in thereagent vessel in the dispensing chip; four nucleic acid substrates canbe added in this dispensing chip in advance, and sealed as it is andfreeze-dried. If a reagent distributor seals and freeze-dries reagentsin advance, investigators only have to load such already sealeddispensing chip into the dispensing head before experiment. Thedispensing chip is not deteriorated by sterilization or freeze-drying,and the manufacturing cost is low. Therefore, dispensing chips designedto be disposable can significantly reduce possible experimental mistakescaused by the contamination of the reagents.

The present invention is utilized in apparatuses analyzing nucleicacids, which is a fundamental tool in life sciences and bioindustries,and, in particular, is applied in DNA sequence determination apparatusesand DNA examination apparatuses.

It should be further understood by those skilled in the art thatalthough the foregoing description has been made on embodiments of theinvention, the invention is not limited thereto and various changes andmodifications may be made without departing from the spirit of theinvention and the scope of the appended claims.

1. An analysis apparatus, wherein the analysis apparatus comprises:reagent container-holding means for holding a reagent containercontaining a reagent; moving means for moving said reagentcontainer-holding means vertically; a reaction vessel for receiving asupply of said reagent from said reagent container and containing theliquid; pressurizing means for applying pressure to said reagentcontainer to supply said reagent therefrom to said reaction vessel;vibrating means for applying vibration to said reaction vessel; and adetector for optical detection for said reaction vessel.
 2. An analysisapparatus, wherein the analysis apparatus comprises: reagentcontainer-holding means for holding a reagent container comprising areagent delivering part and containing a reagent; moving means formoving said reagent container-holding means vertically; a reactionvessel for receiving a supply of said reagent from said reagentcontainer and containing a sample; pressurizing means for applyingpressure to said reagent container to supply said reagent therefrom tosaid reaction vessel; vibrating means for applying vibration to saidreaction vessel; a detector for optical detection for said reactionvessel; and a negative pressure-generating unit for providing an airlayer inside said reagent delivering part.
 3. The analysis apparatusaccording to claim 1, wherein said reagent container comprises a reagentvessel part and a reagent delivering part; and said moving means movessaid reagent container-holding means so that when said reagent issupplied, a delivering tip of said reagent delivering part becomes incontact with said liquid, and after said reagent is supplied, thedelivering tip of said reagent delivering part is separated from saidliquid.
 4. The analysis apparatus according to claim 2, wherein saidreagent container comprises a reagent vessel part and a reagentdelivering part; and said moving means moves said reagentcontainer-holding means so that when said reagent is supplied, adelivering tip of said reagent delivering part becomes in contact withsaid liquid, and after said reagent is supplied, the delivering tip ofsaid reagent delivering part is separated from said liquid.
 5. Theanalysis apparatus according to claim 1, wherein said pressurizing meansapplies pressure by supplying gas; and said reagent container-holdingmeans further comprises a gas flow path connecting said pressurizingmeans and said reagent container.
 6. The analysis apparatus according toclaim 1, wherein said reagent container comprises a first vesselcontaining a first liquid, a second vessel containing a second liquid, athird vessel containing a third liquid, and a fourth vessel containing afourth liquid; and said pressurizing means applies pressure to supplyany one of said first liquid, said second liquid, said third liquid, andsaid fourth liquid to said reaction vessel.
 7. The analysis apparatusaccording to claim 2, wherein said reagent container comprises a firstvessel containing a first liquid, a second vessel containing a secondliquid, a third vessel containing a third liquid, and a fourth vesselcontaining a fourth liquid; and said pressurizing means applies pressureto supply any one of said first liquid, said second liquid, said thirdliquid, and said fourth liquid to said reaction vessel.
 8. The analysisapparatus according to claim 1, wherein said reagent container comprisesa first vessel containing a first liquid, a second vessel containing asecond liquid, a third vessel containing a third liquid, and a fourthvessel containing a fourth liquid; and said pressurizing means appliespressure to supply any two or more of said first liquid, said secondliquid, said third liquid, and said fourth liquid to said reactionvessel.
 9. The analysis apparatus according to claim 2, wherein saidreagent container comprises a first vessel containing a first liquid, asecond vessel containing a second liquid, a third vessel containing athird liquid, and a fourth vessel containing a fourth liquid; and saidpressurizing means applies pressure to supply any two or more of saidfirst liquid, said second liquid, said third liquid, and said fourthliquid to said reaction vessel.
 10. The analysis apparatus according toclaim 1, wherein said reagent container-holding means holds a pluralityof said reagent containers at intervals of 9 mm.
 11. The analysisapparatus according to claim 2, wherein said reagent container-holdingmeans holds a plurality of said reagent containers at intervals of 9 mm.12. The analysis apparatus according to claim 2, wherein said negativepressure-generating unit is a microejector.
 13. The analysis apparatusaccording to claim 2, wherein said negative pressure-generating unitcomprises a narrow tube and a housing linked to said pressurizing means.14-18. (canceled)